CN116445533A - Pichia pastoris engineering bacteria for preparing high-expression nano antibody and rapid screening method - Google Patents
Pichia pastoris engineering bacteria for preparing high-expression nano antibody and rapid screening method Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention discloses a method for high-expression of nano antibody Pichia pastoris engineering bacteria, which comprises the following steps: construction, amplification, linearization and recovery of the nano expression plasmid, culturing of the monoclonal, and transformation into the recipient cell. The invention provides a pichia pastoris engineering strain which does not use the concentration of antibiotics and can rapidly screen high expression of nano-antibodies.
Description
Technical Field
The invention belongs to the technical fields of bioengineering and medical engineering, and particularly relates to a method for preparing high-expression nano antibody Pichia pastoris engineering bacteria and rapidly screening.
Background
Nanobodies were first reported by belgium scientists in 1993 in the journal of Nature as single domain antibodies consisting of the variable region of natural heavy chain antibodies from which the light chain was deleted in camelids (camels, llamas, alpacas and closely related species), which antibodies comprise only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, the VHH crystals being 2.5nm long and 4nm long and having a molecular weight of only 15kDa, and are therefore called nanobodies. Nanobodies (Nbs) are novel members of antibody families, have the advantages of small relative molecular mass, simple humanization, high affinity, high stability, microbial expression, low immunogenicity, strong penetrability, good solubility and the like, and are of great interest in medical basic research and disease diagnosis and treatment.
Nanobodies are superior to traditional antibodies in many respects. The special structure of the VHH single-domain antibody based on alpaca heavy chain antibody has the advantages of the traditional antibody and small molecular drugs, almost perfectly overcomes the defects of long development period, lower stability, harsh preservation conditions and the like of the traditional antibody, and gradually becomes an emerging force in new generation therapeutic biological medicine and clinical diagnostic reagents. The advantages of nanobodies compared to conventional antibodies are: 1. the molecular weight is small, and the blood brain barrier can be penetrated; 2. high expression in prokaryotic or eukaryotic systems; 3. the specificity is strong, and the affinity is high; 4. has weak immunogenicity to human. The application advantages of the nano antibody are used for biological medicine research and development (genetic engineering medicine research and development, ADC medicine research and development); is used for clinical in vitro diagnosis (colloidal gold method, enzyme-linked immunosorbent method, electrochemiluminescence method); is used for basic researches such as tumor research, immunological research and the like.
Expression systems commonly used for nanobodies are prokaryotic expression systems, eukaryotic expression systems and mammalian cell expression systems. The prokaryotic expression operation using the escherichia coli as a main host is relatively simple, the yield is high, and the production cost is relatively low. However, the expressed protein is not modified, the nano antibody protein obtained by cytoplasmic expression often exists in the form of inclusion bodies, and the later preparation of the active nano antibody by inclusion body renaturation is still difficult. Although the mammalian cells are suitable for the expression production of the nanobody, the production cost of the nanobody is far higher than that of the microorganism, and meanwhile, the production period is relatively longer and the requirements on equipment and personnel are higher. The yeast expression system is between the two, so that the yeast expression system can not only express the exogenous protein which is soluble, correctly folded and has simple glycosylation, but also secrete the exogenous protein into a culture medium, thereby facilitating the later purification step. There are many reports on the expression of nanobodies or foreign proteins by using pichia pastoris as engineering bacteria, but the rapid acquisition of stable and high-expression pichia pastoris engineering bacteria is an essential link in industrial production. In industrial production, all the obtained pichia pastoris engineering bacteria are often expressed and screened by a shake flask, and although the process can finally obtain the high-expression pichia pastoris engineering bacteria, a large amount of manpower and material resources are wasted in the process, so that the screening efficiency is low. In the field of expression of nanobodies by yeast cells, pichia pastoris engineering bacteria to obtain high expression are dependent on a plurality of factors, however, it is generally considered that for Pichia pastoris, high-copy gene cloning corresponds to related high-expression nanobodies, screening Pichia pastoris engineering bacteria with different copy numbers by using different antibiotic concentrations is a relatively simple and efficient screening method, and the method can be used for rapidly screening the Pichia pastoris engineering bacteria with high expression of nanobodies on a high antibiotic flat plate.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide the engineering bacteria for preparing the high-expression nano antibody Pichia pastoris.
In order to solve the technical problems, the invention provides the following technical scheme: a method for preparing high-expression nano antibody Pichia pastoris engineering bacteria comprises the following steps:
construction, amplification, linearization and recovery of nano expression plasmids: optimizing the nanobody genes to obtain optimized nanobody genes; extracting original plasmid, amplifying the plasmid, and performing enzyme digestion on the amplified plasmid to prepare a linearization nano expression plasmid;
culturing the monoclonal: 1. Mu.L of the recipient bacteria was streaked on a plate containing the medium. Inverted in a constant temperature incubator, and cultivated at constant temperature until dispersed monoclonal colonies grow out; the monoclonal colony is picked up and inoculated into 5mL of culture medium, and the culture is carried out at a constant temperature of 250 rpm. The culture is inoculated into fresh culture medium, and is cultivated at constant temperature of 250rpm until the OD600 = 1.3-1.5; collecting the culture, centrifuging to remove supernatant, and pre-cooling sterile water to resuspend cells; centrifuging to remove supernatant, and pre-cooling the sterilized water to resuspend cells; centrifuging to remove supernatant, pre-cooling LiAc preparation liquid, re-suspending cells, and standing; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; sub-packaging competent cells to be used in subsequent experiments;
transformation into recipient cells: adding precooled linearization recombinant plasmid into recipient bacterium competent cells, mixing uniformly, and ice-bathing; transferring the mixed solution into a precooled electric rotating cup, and placing the electric rotating cup into an electric rotating instrument (Bio-Rad); the parameters of the electrotransfer instrument are set to be Pichia preset, and the electric shock is immediately cooled for 30 seconds in an ice bath after 1 time of electric shock, and the electric shock is more than or equal to 5 times; adding sorbitol solution, gently blowing and beating uniformly, and transferring the content into a sterile centrifuge tube; centrifuging for 5 minutes, removing part of supernatant, and carrying out constant-temperature culture while leaving 200 mu L of bacterial liquid to resuspend cells; the obtained cell suspension is coated on a flat plate containing a culture medium of selective antibiotics, and the rest suspension is coated on a non-resistant flat plate; standing at room temperature until no significant flowing liquid is on the plate, transferring to a constant temperature incubator, and culturing upside down until a selective antibiotic plate grows obvious monoclonal colonies;
a method of selecting a positive copy strain; after the transformation, a significant monoclonal colony grows on a low-concentration selective antibiotic resistance plate, the monoclonal colony is numbered, and meanwhile, the monoclonal colony is transferred to a resistance plate with gradient antibiotic concentration, and a mark is made; inverting the plate after the plate rotation until the culture plate grows obvious monoclonal colonies; the serial numbers of colonies growing on plates with different antibiotic concentrations are recorded, and monoclonal strains growing on plates with different antibiotic concentrations are picked for expression verification.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the recipient bacterium is P.pastoris X-33 glycerol bacterium.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the constant temperature is kept at 25-30 ℃.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the culture medium is YPD culture medium.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the selective antibiotic is Zeocin.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the amino acid sequence of the prepared monovalent nano antibody is shown as SEQ 1, and the corresponding DNA sequence is shown as SEQ 2.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the amino acid sequence of the prepared bivalent nano antibody is shown as SEQ 3, and the corresponding DNA sequence is shown as SEQ 4.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the amino acid sequence of the prepared trivalent nanometer antibody is shown as SEQ 5, and the corresponding DNA sequence is shown as SEQ 6.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the transformant growing on the high-concentration flat plate is the high-expression nano antibody engineering bacteria.
As a preferable scheme of the method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, the invention provides a method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria, wherein: the engineering bacteria are pichia pastoris.
The invention has the beneficial effects that:
there are many reports on the expression of nanobodies or foreign proteins by using pichia pastoris as engineering bacteria, but the rapid acquisition of stable and high-expression pichia pastoris engineering bacteria is an essential link in industrial production. In industrial production, all the obtained pichia pastoris engineering bacteria are often expressed and screened by a shake flask, and although the process can finally obtain the high-expression pichia pastoris engineering bacteria, a large amount of manpower and material resources are wasted in the process, so that the screening efficiency is low. In the field of expression of nanobodies by yeast cells, pichia pastoris engineering bacteria to obtain high expression are dependent on a plurality of factors, however, it is generally considered that for Pichia pastoris, high-copy gene cloning corresponds to related high-expression nanobodies, screening Pichia pastoris engineering bacteria with different copy numbers by using different antibiotic concentrations is a relatively simple and efficient screening method, and the method can be used for rapidly screening the Pichia pastoris engineering bacteria with high expression of nanobodies on a high antibiotic flat plate.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a graph showing expression plasmids of monovalent nanobodies prepared in the examples of the present invention;
FIG. 2 shows a linearized monovalent nanobody expression plasmid prepared in the examples of the present invention;
FIG. 3 shows the screening of high-resistance Pichia pastoris expression engineering bacteria at different antibiotic concentrations prepared in the examples of the present invention;
FIG. 4 shows the verification of monovalent nanobodies expressed by Pichia pastoris engineering bacteria with different concentrations of resistance prepared in the embodiment of the invention;
FIG. 5 is a graph showing statistics of expression amounts of monovalent nanobodies expressed by Pichia pastoris engineering bacteria with different resistance concentrations prepared in the embodiment of the invention;
FIG. 6 is a graph showing expression plasmids of bivalent nanobodies prepared in the examples of the present invention;
FIG. 7 shows a linearized bivalent nanobody expression plasmid prepared in the examples of the present invention;
FIG. 8 shows the screening of high-resistance Pichia pastoris expression engineering bacteria at different antibiotic concentrations prepared in the examples of the present invention;
FIG. 9 shows the validation of the expression of bivalent nanobody by pichia pastoris engineering bacteria with different concentrations of resistance prepared in the examples of the present invention;
FIG. 10 shows statistics of expression amounts of the divalent nanobodies expressed by the Pichia pastoris engineering bacteria with different resistance concentrations prepared in the embodiment of the invention;
FIG. 11 is a diagram of a trivalent nanobody expression plasmid prepared in the examples of the present invention;
FIG. 12 shows a linearized trivalent nanobody expression plasmid prepared in the examples of the present invention;
FIG. 13 shows the screening of Pichia pastoris expression engineering bacteria with high resistance at different antibiotic concentrations prepared in the examples of the present invention;
FIG. 14 shows the verification of the expression of trivalent nanobodies by Pichia pastoris engineering bacteria with different concentrations of resistance prepared in the examples of the invention;
FIG. 15 shows statistics of expression amounts of trivalent nanobodies expressed by Pichia pastoris engineering bacteria with different resistance concentrations prepared in the examples of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1
Construction of monovalent nanobody expression plasmids. The codon of the monovalent nanobody gene sequence is optimized to be a codon favored by pichia pastoris, and the optimized sequence needs to avoid the SacI enzyme cutting site. And (3) transferring the optimized sequence to a gene synthesis company, synthesizing an insertion Sequence (SI), integrating the synthesized sequence on a cloning vector, wherein the common cloning vector is pPICZ alpha A, and finally constructing a pPICZ alpha A-monovalent nanobody expression plasmid, wherein the expression plasmid map is shown in figure 1.
Monovalent nanobody expression plasmid amplification, after the strain construction was successful, plasmid amplification was performed and 10 μl of glycerol bacteria was inoculated into 10mL of LB medium (50 mL centrifuge tube) or 100mL of LB medium (500 mL shake flask). Placing into a constant temperature shaking table, culturing at 220rpm and 37 ℃ for 16h.
Extraction of monovalent nanobody plasmids. The plasmids were extracted using SanPrep column type plasmid DNA miniprep kit (Shanghai) or endotoxin-free plasmid macroextraction kit (enhanced, centrifugal column, tiangen Biochemical technology (Beijing) Co., ltd.) and the extraction process was referred to the product specifications.
Linearization of monovalent nanobody recombinant plasmids. The recombinant plasmid pPICZαA-monovalent nanobody was digested with SacI (QuickCutSacI, taKaRa) and linearized, the linearization results of which are shown in FIG. 2. The linearized recombinant plasmid pPICZ alpha A-monovalent nanobody was purified and recovered using SanPrep column PCR product kit (Shanghai Co., ltd.).
mu.L of P.pastoris X-33 glycerol bacteria were streaked onto YPD plates. Inverted in a constant temperature incubator, and cultured at 25-30 ℃ until dispersed monoclonal colonies (36-48 h) are grown. The monoclonal colonies were picked up and inoculated with 5mL of YPD medium (50 mL centrifuge tube) and placed in a thermostatic shaker at 25-30℃and 250rpm for 24 hours. 100. Mu.L of the culture was inoculated with 100mL of fresh YPD medium (500 mL Erlenmeyer flask), and incubated at 25-30℃in a thermostatic shaker at 250rpm for 24h until OD600 = 1.3-1.5. 100mL of the culture was collected, centrifuged at 1500 Xg at 4℃for 5 minutes, the supernatant was removed, and the cells were resuspended in 100mL of pre-chilled sterile water. The cells were resuspended in 100mL of pre-chilled sterile water by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed. The cells were resuspended in 10mL of pre-chilled LiAc preparation (10 mM Tris-HCl, 100mM LiAc, 10mM DTT, 0.6M Sorbitol;pH 7.5) by centrifugation at 1500 Xg for 5 min at 4℃and allowed to stand at 4℃for 30 min. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 4mL of pre-chilled 1M sorbitol solution. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 2mL of pre-chilled 1M sorbitol solution. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 1mL of pre-chilled 1M sorbitol solution. The cells were resuspended in 100. Mu.L of pre-chilled 1M sorbitol solution by centrifugation at 1500 Xg for 5 min at 4 ℃. Competent cells were sub-packaged, 80 μl each, and stored on ice for later experiments.
80 mu L of P.P astoris X-33 competent cells are added with 5-10 mu g of precooled linear monovalent nanometer antibody recombinant plasmid (less than or equal to 10 mu L), and the mixture is uniformly mixed and ice-bathed for 5 minutes. The mixture was transferred to a pre-chilled electrocuvette, which was placed in an electrotometer (Bio-Rad). Parameters of the electrotometer are set to be Pichia preset, and the electrotometer is immediately cooled for 30 seconds in an ice bath after electric shock for 1 time, and the electric shock is more than or equal to 5 times. 1mL of pre-chilled 1M sorbitol solution was added, gently swirled to homogeneity, and the contents transferred to a sterile centrifuge tube. Centrifugation at 1500 Xg for 5 min, partial supernatant was removed, 200. Mu.L of the bacterial suspension was left to resuspend the cells, and the cells were incubated at 30℃for 1.5 hours. A Zeocin resistant (100. Mu.g/mL) YPD plate was plated with 100. Mu.L of the cell suspension, and the remaining suspension was plated with YPD non-resistant plate (control). Standing at room temperature until no significant flowing liquid is present on the plate, transferring to a constant temperature incubator, and inversely culturing at 25-30 ℃ for 48-72 hours until the Zeocin-resistant YPD plate grows obvious monoclonal colonies.
And (5) screening the monovalent nano antibody high-copy Pichia pastoris engineering strain. After transformation, obvious monoclonal colonies were grown on low concentration Zeocin resistant YPD plates, and the monoclonal colonies were numbered while transferred to different antibiotic concentration Zeocin resistant YPD plates (100. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL, 2000. Mu.g/mL) and labeled. The plate after the plate transfer is inversely cultured for 24-48 hours at the temperature of 25-30 ℃ and obvious monoclonal colonies are grown on the plate. The serial numbers of colonies growing on plates with different antibiotic concentrations are recorded, and monoclonal strains growing on plates with different antibiotic concentrations are picked for expression verification. The results of the different antibiotic concentrations are shown in FIG. 3.
The monovalent nanobody is expressed by the pichia pastoris engineering bacteria in an induction way. Monoclonal colonies of P.Pastois X-33/pPICZ alpha A-monovalent nanobodies on plates with different antibiotic concentrations were picked and inoculated with 5mL BMGY (50 mL centrifuge tube) respectively. Placing into a constant temperature shaking table, and culturing at 25-30deg.C and 250rpm for 16 hr. The OD600 of the culture was measured, and a 10OD 600. Sup. Th cell culture was collected. 1,500 Xg was centrifuged for 5 minutes, the supernatant was removed, and the cells were resuspended in 10mL BMMY medium. Placing into a constant temperature shaking table, culturing at 30deg.C and 250rpm for 72 hr. Every 24 hours of incubation, 50 μl methanol (0.5%) was replenished to each centrifuge tube. After completion of the incubation, 10,000Xg was centrifuged for 5 minutes, and the supernatant was collected.
Identification and quantification of monovalent nanobodies. The sample and protein Loading Buffer are mixed in proportion so that the concentration of the protein Loading Buffer in the final sample is not less than 1X, and the final sample is denatured by boiling water bath at 98 ℃ for 10min. Subsequently, an electrophoresis tank and a rubber plate (sealing-removing adhesive tape) are installed, electrophoresis buffer solution is poured into the electrophoresis tank, the inner tank should overflow, and the liquid level of the outer tank should be higher than that of the electrode wire. Slowly removing the comb of the prefabricated glue, taking the needle cylinder to absorb the buffer solution, and gradually extending into the glue holes to blow off the glycerol. Closing the cell cover, electrifying, and electrophoresis for 40 minutes under 80V voltage until the sample is compressed into a line. The electrophoresis was continued by increasing the voltage value by 120V until the indicator tape was near or reached the bottom of the gel. Dyeing and decoloring the gel after electrophoresis by using a protein dyeing instrument, and setting the program to be Stain 3min; destin 2min; destin 1min. The gel is transferred into a gel imaging system, imaged, image modified, lanes arranged, lanes marked and corresponding analysis is performed. The SDS-PAGE result of the monoclonal strain supernatant of the Pichia pastoris engineering bacteria screened by different antibiotic concentrations is shown in figure 4, the expressed protein quantity is measured by using software, and the monovalent nanobody expression quantity of the monoclonal strain screened by the flat plates with different antibiotic resistance concentrations is shown in figure 5.
Example 2
Construction of bivalent nanobody expression plasmids. The codon of the bivalent nano antibody gene sequence is optimized to be a codon favored by pichia pastoris, and the optimized sequence needs to avoid the SacI enzyme cutting site. And (3) feeding the optimized sequence to a gene synthesis company, synthesizing an insertion Sequence (SI), integrating the synthesized sequence on a cloning vector, wherein the common cloning vector is pPICZ alpha A, and finally constructing a pPICZ alpha A-bivalent nanobody expression plasmid, wherein the expression plasmid map is shown in figure 6.
The bivalent nanobody expression plasmid is amplified, and after the strain is successfully constructed, the plasmid is amplified, and 10 mu L of glycerinum is added into 10mL of LB culture medium (50 mL centrifuge tube) or 100mLLB culture medium (500 mL shake flask). Placing into a constant temperature shaking table, culturing at 220rpm and 37 ℃ for 16 hours.
Extraction of bivalent nanobody plasmids. The plasmids were extracted using SanPrep column type plasmid DNA miniprep kit (Shanghai) or endotoxin-free plasmid macroextraction kit (enhanced, centrifugal column, tiangen Biochemical technology (Beijing) Co., ltd.) and the extraction process was referred to the product specifications.
Linearization of bivalent nanobody recombinant plasmids. The recombinant plasmid pPICZαA-bivalent nanobody was digested with SacI (QuickCutSacI, taKaRa) and linearized, and the linearization results are shown in FIG. 7. The linearized recombinant plasmid pPICZ alpha A-bivalent nanobody was purified and recovered by using SanPrep column PCR product kit (Shanghai Co., ltd.).
mu.L of P.pastoris X-33 glycerol bacteria were streaked onto YPD plates. Inverted in a constant temperature incubator, and cultured at 25-30 ℃ until dispersed monoclonal colonies are grown (36-48 hours). The monoclonal colonies were picked up and inoculated with 5mL of YPD medium (50 mL centrifuge tube) and placed in a thermostatic shaker at 25-30℃and 250rpm for 24 hours. 100. Mu.L of the culture was inoculated with 100mL of fresh YPD medium (500 mL Erlenmeyer flask), and incubated at 25-30℃in a thermostatic shaker at 250rpm for 24 hours until OD600 = 1.3-1.5. 100mL of the culture was collected, centrifuged at 1500 Xg at 4℃for 5 minutes, the supernatant was removed, and the cells were resuspended in 100mL of pre-chilled sterile water. The cells were resuspended in 100mL of pre-chilled sterile water by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed. The cells were resuspended in 10mL of pre-chilled LiAc preparation (10 mM Tris-HCl, 100mM LiAc, 10mM DTT, 0.6M Sorbitol;pH 7.5) by centrifugation at 1500 Xg for 5 min at 4℃and allowed to stand at 4℃for 30 min. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 4mL of pre-chilled 1M sorbitol solution. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 2mL of pre-chilled 1M sorbitol solution. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 1mL of pre-chilled 1M sorbitol solution. The cells were resuspended in 100. Mu.L of pre-chilled 1M sorbitol solution by centrifugation at 1500 Xg for 5 min at 4 ℃. Competent cells were sub-packaged, 80 μl each, and stored on ice for later experiments.
80 mu L of P.P astoris X-33 competent cells are added with 5-10 mu g of precooled linearized bivalent nano antibody recombinant plasmid (less than or equal to 10 mu L), and the mixture is uniformly mixed and ice-bathed for 5 minutes. The mixture was transferred to a pre-chilled electrocuvette, which was placed in an electrotometer (BioRad). The parameters of the electrotransfer instrument are set to be Pichia preset, and the electrotransfer instrument is immediately cooled for 30 seconds in an ice bath after electric shock for 1 time, and the electric shock is more than or equal to 5 times. 1mL of pre-chilled 1M sorbitol solution was added, gently swirled to homogeneity, and the contents transferred to a sterile centrifuge tube. Centrifugation at 1500 Xg for 5 min, partial supernatant was removed, 200. Mu.L of the bacterial suspension was left to resuspend the cells, and the cells were incubated at 30℃for 1.5 hours. A Zeocin resistant (100. Mu.g/mL) YPD plate was plated with 100. Mu.L of the cell suspension, and the remaining suspension was plated with YPD non-resistant plate (control). The culture medium is kept at room temperature until no significant flowing liquid exists on the plate, and is transferred to a constant temperature incubator, and is inversely cultured at 25-30 ℃ until a significant monoclonal colony is grown on the Zeocin-resistant YPD plate.
And (5) screening bivalent nano antibody high copy strains. After transformation, significant monoclonal colonies were grown on low concentration Zeocin resistant YPD plates, and the monoclonal strains were numbered while transferred to different antibiotic concentration Zeocin resistant YPD plates (100. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL, 2000. Mu.g/mL) and labeled. The plate after the plate transfer is inversely cultured for 24-48 hours at the temperature of 25-30 ℃ and obvious monoclonal colonies are grown on the plate. The serial numbers of colonies growing on plates with different antibiotic concentrations are recorded, and monoclonal strains growing on plates with different antibiotic concentrations are picked for expression verification. The results of the different antibiotic concentrations are shown in FIG. 8.
The bivalent nano antibody is expressed by the pichia pastoris engineering bacteria in an induction way. Monoclonal colonies of P.Pastois X-33/pPICZ alpha A-bivalent nanobody on plates with different antibiotic concentrations were picked and respectively inoculated with 5mL BMGY (50 mL centrifuge tube). Placing into a constant temperature shaking table, and culturing at 25-30deg.C and 250rpm for 16 hr. The OD600 of the culture was measured, and 10OD 600. Mu.mL of the bacterial liquid was collected. 1,500 Xg was centrifuged for 5 minutes, the supernatant was removed, and the cells were resuspended in 10mL BMMY medium. Placing into a constant temperature shaking table, culturing at 30deg.C and 250rpm for 72 hr. Every 24 hours of incubation, 50 μl methanol (0.5%) was replenished to each centrifuge tube. After completion of the incubation, 10,000Xg was centrifuged for 5 minutes, and the supernatant was collected.
Identification and quantification of bivalent nanobodies. The sample and protein Loading Buffer are mixed in proportion so that the concentration of the protein Loading Buffer in the final sample is not less than 1X, and the final sample is denatured by boiling water bath at 98 ℃ for 10min. Subsequently, an electrophoresis tank and a rubber plate (sealing-removing adhesive tape) are installed, electrophoresis buffer solution is poured into the electrophoresis tank, the inner tank should overflow, and the liquid level of the outer tank should be higher than that of the electrode wire. Slowly pulling out the hole-making comb, taking the needle cylinder to absorb the buffer solution, and gradually extending into the glue holes to blow off the glycerol. Closing the cell cover, electrifying, and electrophoresis for 20 minutes under 80V voltage until the sample is compressed into a line. The electrophoresis was continued by increasing the voltage value by 120V until the indicator tape was near or reached the bottom of the gel. Dyeing and decoloring the gel after electrophoresis by using a protein dyeing instrument, and setting the program to be Stain 3min; destin 2min; destin 1min. The gel is transferred into a gel imaging system, imaged, image modified, lanes arranged, lanes marked and corresponding analysis is performed. The SDS-PAGE results of the monoclonal strains of the Pichia pastoris engineering bacteria screened by different antibiotic concentrations are shown in figure 9, the expressed protein quantity is measured by software, and the statistics of the expression quantity of the bivalent nano antibodies of the monoclonal strains screened by the flat plates with different antibiotic resistance concentrations are shown in figure 10.
Example 3
And constructing a trivalent nanometer antibody expression plasmid. The codon of the trivalent nanometer antibody gene sequence is optimized to be a codon favored by pichia pastoris, and the optimized sequence needs to avoid the SacI enzyme cutting site. The optimized sequence is transferred to a gene synthesis company to synthesize an insertion Sequence (SI), the synthesized sequence is integrated on a cloning vector, the common cloning vector is pPICZ alpha A, and finally, the pPICZ alpha A-trivalent nano antibody expression plasmid is constructed, and the expression plasmid map is shown in figure 11.
Trivalent nanobody expression plasmid amplification, after the strain construction was successful, plasmid amplification was performed, and 10. Mu.L of glycerol bacteria were added to 10mL LB medium (50 mL centrifuge tube) or 100mL LLB medium (500 mL shake flask). Placing into a constant temperature shaking table, culturing at 220rpm and 37 ℃ for 16h.
And (3) extracting trivalent nanometer antibody plasmids. The plasmids were extracted using SanPrep column type plasmid DNA miniprep kit (Shanghai) or endotoxin-free plasmid macroextraction kit (enhanced, centrifugal column, tiangen Biochemical technology (Beijing) Co., ltd.) and the extraction process was referred to the product specifications.
Linearization of trivalent nanobody recombinant plasmids. The recombinant plasmid pPICZαA-trivalent nanobody was digested with SacI (Quick Cut SacI, taKaRa) to linearize it, and the linearization results are shown in FIG. 12. The linearized recombinant plasmid pPICZ alpha A-trivalent nanobody was purified and recovered by using SanPrep column PCR product kit (Shanghai Co., ltd.).
mu.L of P.pastoris X-33 glycerol bacteria were streaked onto YPD plates. Inverted in a constant temperature incubator, cultured at 30 ℃ until dispersed monoclonal colonies are grown for 48h. The monoclonal colonies were picked up and inoculated with 5mL of YPD medium (50 mL centrifuge tube) and placed in a thermostatic shaker at 30℃and 250rpm for 24 hours. 100. Mu.L of the culture was inoculated with 100mL of fresh YPD medium (500 mL Erlenmeyer flask), and incubated at 30℃in a thermostatic shaker at 250rpm for 24 hours until OD600 = 1.4. 100mL of the culture was collected, centrifuged at 1500 Xg at 4℃for 5 minutes, the supernatant was removed, and the cells were resuspended in 100mL of pre-chilled sterile water. The cells were resuspended in 100mL of pre-chilled sterile water by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed. The cells were resuspended in 10mL of pre-chilled LiAc preparation (10 mM Tris-HCl, 100mM LiAc, 10mM DTT, 0.6M Sorbitol;pH 7.5) by centrifugation at 1500 Xg for 5 min at 4℃and allowed to stand at 4℃for 30 min. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 4mL of pre-chilled 1M sorbitol solution. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 2mL of pre-chilled 1M sorbitol solution. The cells were resuspended by centrifugation at 1500 Xg for 5 minutes at 4℃and the supernatant removed and 1mL of pre-chilled 1M sorbitol solution. The cells were resuspended in 100. Mu.L of pre-chilled 1M sorbitol solution by centrifugation at 1500 Xg for 5 min at 4 ℃. Competent cells were sub-packaged, 80 μl each, and stored on ice for later experiments.
80 mu L of P.P astoris X-33 competent cells are added with 10 mu g of precooled linearized trivalent nanometer antibody recombinant plasmid (less than or equal to 10 mu L), and the mixture is uniformly mixed and ice-bathed for 5 minutes. The mixture was transferred to a pre-chilled electrocuvette, which was placed in an electrotometer (Bio-Rad). The parameters of the electrotransfer instrument are set to be Pichia preset, and the electrotransfer instrument is immediately cooled for 30 seconds in an ice bath after electric shock for 1 time, and the electric shock is more than or equal to 5 times. 1mL of pre-chilled 1M sorbitol solution was added, gently swirled to homogeneity, and the contents transferred to a sterile centrifuge tube. Centrifugation at 1500 Xg for 5 min, partial supernatant was removed, 200. Mu.L of the bacterial suspension was left to resuspend the cells, and the cells were incubated at 30℃for 1.5 hours. A Zeocin resistant (100. Mu.g/mL) YPD plate was plated with 100. Mu.L of the cell suspension, and the remaining suspension was plated with YPD non-resistant plate (control). The culture medium is kept at room temperature until no significant flowing liquid exists on the plate, and is transferred to a constant temperature incubator, and is inversely cultured for 72 hours at 30 ℃ until a significant monoclonal colony is grown on the Zeocin-resistant YPD plate.
And (5) screening the trivalent nanometer antibody high-copy Pichia pastoris engineering strain. After transformation, obvious monoclonal colonies were grown on low concentration Zeocin resistant YPD plates, and the monoclonal colonies were numbered while transferred to different antibiotic concentration Zeocin resistant YPD plates (100. Mu.g/mL, 500. Mu.g/mL, 1000. Mu.g/mL, 2000. Mu.g/mL) and labeled. The plate after plate transfer was cultured upside down at 30℃for 48 hours, and the plate developed obvious monoclonal colonies. The serial numbers of colonies growing on plates with different antibiotic concentrations are recorded, and monoclonal strains growing on plates with different antibiotic concentrations are picked for expression verification. The results of the different antibiotic concentrations are shown in FIG. 13.
The trivalent nanometer antibody is expressed by the pichia pastoris engineering bacteria in an induction way. Monoclonal colonies of P.Pastois X-33/pPICZ alpha A-trivalent nanobodies on plates with different antibiotic concentrations were picked and inoculated into 5mLBMGY (50 mL centrifuge tube) respectively. Placing into a constant temperature shaking table, culturing at 30deg.C and 250rpm for 16 hr. The OD600 of the culture was measured, and 10OD 600. Mu.mL of the bacterial liquid was collected. The cells were resuspended in 10mLBMMY medium by centrifugation at 1,500 Xg for 5 minutes and the supernatant removed. Placing into a constant temperature shaking table, culturing at 30deg.C and 250rpm for 72 hr. Every 24 hours of incubation, 50 μl methanol (0.5%) was replenished to each centrifuge tube. After completion of the incubation, 10,000Xg was centrifuged for 5 minutes, and the supernatant was collected.
Identification and quantification of trivalent nanobodies. The sample and protein Loading Buffer are mixed in proportion so that the concentration of the protein Loading Buffer in the final sample is not less than 1X, and the final sample is denatured by boiling water bath at 98 ℃ for 10min. Subsequently, an electrophoresis tank and a rubber plate (sealing-removing adhesive tape) are installed, electrophoresis buffer solution is poured into the electrophoresis tank, the inner tank should overflow, and the liquid level of the outer tank should be higher than that of the electrode wire. Slowly pulling out the hole-making comb, taking the needle cylinder to absorb the buffer solution, and gradually extending into the glue holes to blow off the glycerol. Closing the cell cover, electrifying, and carrying out electrophoresis for 40min under 80V voltage until the sample is compressed into a line. The electrophoresis was continued by increasing the voltage value by 120V until the indicator tape was near or reached the bottom of the gel. Dyeing and decoloring the gel after electrophoresis by using a protein dyeing instrument, and setting the program to be Stain 3min; destin 2min; destin 1min. The gel is transferred into a gel imaging system, imaged, image modified, lanes arranged, lanes marked and corresponding analysis is performed. The SDS-PAGE result of the monoclonal strain supernatant of the Pichia pastoris engineering bacteria screened by different antibiotic concentrations is shown in figure 14, the expressed protein quantity is measured by software, and the expression quantity of the monoclonal strain trivalent nanometer antibody screened by the flat plates with different antibiotic resistance concentrations is shown in figure 15 and is high in concentration.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. A method for preparing high-expression nano antibody Pichia pastoris engineering bacteria is characterized in that: the method comprises the following steps:
construction, amplification, linearization and recovery of nano expression plasmids: optimizing the nanobody genes to obtain optimized nanobody genes; extracting original plasmid, amplifying the plasmid, and performing enzyme digestion on the amplified plasmid to prepare a linearization nano expression plasmid;
culturing the monoclonal: 1. Mu.L of the recipient bacteria was streaked on a plate containing the medium. Inverted in a constant temperature incubator, and cultivated at constant temperature until dispersed monoclonal colonies grow out; the monoclonal colony is picked up and inoculated into 5mL of culture medium, and the culture is carried out at a constant temperature of 250 rpm. The culture is inoculated into fresh culture medium, and is cultivated at constant temperature of 250rpm until the OD600 = 1.3-1.5; collecting the culture, centrifuging to remove supernatant, and pre-cooling sterile water to resuspend cells; centrifuging to remove supernatant, and pre-cooling the sterilized water to resuspend cells; centrifuging to remove supernatant, pre-cooling LiAc preparation liquid, re-suspending cells, and standing; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; centrifuging to remove the supernatant, and pre-cooling the sorbitol solution to resuspend the cells; sub-packaging competent cells to be used in subsequent experiments;
transformation into recipient cells: adding precooled linearization recombinant plasmid into recipient bacterium competent cells, mixing uniformly, and ice-bathing; transferring the mixed solution into a precooled electric rotating cup, and placing the electric rotating cup into an electric rotating instrument (Bio-Rad); the parameters of the electrotransfer instrument are set to be Pichia preset, and the electric shock is immediately cooled for 30 seconds in an ice bath after 1 time of electric shock, and the electric shock is more than or equal to 5 times; adding sorbitol solution, gently blowing and beating uniformly, and transferring the content into a sterile centrifuge tube; centrifuging for 5 minutes, removing part of supernatant, and carrying out constant-temperature culture while leaving 200 mu L of bacterial liquid to resuspend cells; the obtained cell suspension is coated on a flat plate containing a culture medium of selective antibiotics, and the rest suspension is coated on a non-resistant flat plate; standing at room temperature until no significant flowing liquid is on the plate, transferring to a constant temperature incubator, and culturing upside down until a selective antibiotic plate grows obvious monoclonal colonies;
a method for simply and quickly selecting positive copy strains; after the transformation, a significant monoclonal colony grows on a low-concentration selective antibiotic resistance plate, the monoclonal colony is numbered, and meanwhile, the monoclonal colony is transferred to a resistance plate with gradient antibiotic concentration, and a mark is made; inverting the plate after the plate rotation until the culture plate grows obvious monoclonal colonies; the serial numbers of colonies growing on plates with different antibiotic concentrations are recorded, and monoclonal strains growing on plates with different antibiotic concentrations are picked for expression verification.
2. The method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria according to claim 1, wherein the method comprises the following steps: the recipient bacterium is P.pastoris X-33 glycerol bacterium.
3. The method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria according to claim 1, wherein the method comprises the following steps: the constant temperature is kept at 25-30 ℃.
4. The method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria according to claim 1, wherein the method comprises the following steps: the culture medium is YPD culture medium.
5. The method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria according to claim 1, wherein the method comprises the following steps: the selective antibiotic is Zeocin.
6. The method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria according to claim 1, wherein the method comprises the following steps: the amino acid sequence of the prepared monovalent nano antibody is shown as SEQ 1, and the corresponding DNA sequence is shown as SEQ 2.
7. The method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria according to claim 1, wherein the method comprises the following steps: the amino acid sequence of the prepared bivalent nano antibody is shown as SEQ 3, and the corresponding DNA sequence is shown as SEQ 4.
8. The method for rapidly screening the large molecular weight human collagen pichia pastoris engineering bacteria according to claim 1, wherein the method comprises the following steps: the amino acid sequence of the prepared trivalent nanometer antibody is shown as SEQ 5, and the corresponding DNA sequence is shown as SEQ 6.
9. A method for rapidly screening high-expression nano antibody Pichia pastoris engineering bacteria is characterized in that: the transformant growing on the high-concentration flat plate is the high-expression nano antibody engineering bacteria.
10. The method for rapidly screening pichia pastoris engineering bacteria according to claim 9, wherein the method comprises the following steps: the engineering bacteria are pichia pastoris.
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| CN107686520A (en) * | 2016-08-04 | 2018-02-13 | 信达生物制药(苏州)有限公司 | Anti- PD L1 nano antibodies and its application |
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